The present invention relates to methods for selecting specific ligand and receptor encoding sequences and to kits for carrying out the methods.
There is a continuing need for highly efficient selection systems in the screening of protein libraries, such as antibody libraries. Current systems are based on the display of antibodies on the surface of microorganisms containing the gene of the antibody. Specific clones can then be selected with immobilized antigens, for instance by panning on microtiter plates (Parmley and Smith, 1988, Gene, 73, 305-318 and Barbas III et al, 1991, PNAS 88, 7978-7982), selection on magnetic beads (Hawkins et al, 1992, J. Mol. Biol. 226, 889-896), immunotubes (Marks et al, 1991, J. Mol. Biol, 222, 581-597), affinity chromatography (McCafferty et al, 1990, Nature, 348, 552-554), fluorescence assisted cell sorter (FACS), antigen specific precipitation (Kang et al, 1991, PNAS, 88, 4363-4366) and SAP (Duexc3x1as and Borrebaeck, 1994, Bio/Technology 12, 999-1002).
Several antibody libraries have been constructed on the surface of phages, e.g. a bacteriophage such as fd (McCafferty et al, 1990, Nature, 348, 552-554) or M13 (Barbas III et al, 1991, PNAS, 88, 7978-7982). The possibility of expressing antibodies (scFv) on the surface of bacteria has also been demonstrated by fusions to bacterial membrane proteins like Lpp-Omp A (Francisco et al, 1993, PNAS, 90, 10444-10448) and PAL (Fuchs et al, 1991, Bio/Technology, 9, 1369-1372). For a recent review of antibody display systems and the screening of antibody libraries, see Little, 1994, Biotech. Adv., 12, 539-555.
Antigen libraries have been constructed following essentially the same principles as antibody libraries, e.g. peptide libraries on the surface of bacteriophages (Smith, 1985, Science, 228, 1315-1317). Expression of antigens on the surface of bacteria has been demonstrated by fusions to LamB (Charbit et al, 1988, Gene, 70, 181-189 and Bradbury et al, 1993, Bio/Technology, 1565-1568), Omp A (Pistor and Hoborn, 1989, Klin. Wochenschr., 66, 110-116), fimbriae (Hedegaard and Klemm, 1989, Gene, 85, 115-124 and Hofnung, 1991, Methods Cell Biol., 34, 77-105), IgA protease xcex2 domain (Klauser et al, 1990, EMBO J., 9, 1991-1999) and flagellae (Newton et al, 1989, Science, 244, 70-72).
However, many of the prior art selection steps, such as panning and affinity chromatography, are not very efficient, and even if the yield of antibody or antigen is reasonable, these techniques do not provide any information about the nucleic acid sequence encoding it.
In the past, it has not been possible to combine a ligand library with a receptor library in order not only to clone and select one of the specific binding pair members and their corresponding nucleic acid sequences encoding them, but actually both.
The present invention provides an efficient screening technique to obtain corresponding ligand/receptor molecules and establishes a physical and logical connection between the two and their encoding sequences. This has the advantage that it is simple and rapid and opens up possibilities of applications, such as detecting new ligands and/or receptors, where both are unknown, as well as improvements regarding epitope mapping, localization of gene products and drug design.
Accordingly, in one aspect, the present invention provides a method for selecting nucleic acid sequences encoding ligand and receptor molecules capable of specific binding to each other, the method comprising:
(a) expressing in a host microorganism nucleic acid encoding a surface molecule which is operably linked to the expression of nucleic acid encoding a ligand or receptor molecule, or functional fragments thereof, so that the microorganism expresses the surface molecule and the ligand or receptor molecule as a fusion and displays it on its surface, wherein the host microorganism is modified so that it does not display the surface molecule other than as a fusion with the ligand or receptor molecule;
(b) contacting the modified host microorganism of step (a) with one or more replicable genetic units capable of expressing nucleic acid encoding ligand or receptor molecules, or functional fragments thereof, the ligand or receptor molecules being candidates for specific binding to the molecules displayed by the host microorganisms of step (a) and being expressed as fusions with a surface protein of the replicable genetic unit, wherein binding of the surface displayed ligand and receptor molecules mediates the transfer of the nucleic acid encoding the ligand or receptor from the replicable genetic unit to the microorganism; and,
(c) selecting the host microorganisms containing the nucleic acid sequences encoding the ligand and receptor molecules, or functional fragments thereof.
Thus, in this aspect, the invention provides a method for selecting nucleic acid sequences encoding ligand and receptor molecules, the selection arising from modification to the host microorganism so that infection or transfer of the nucleic acid from the replicable genetic unit takes place when binding of the receptor and ligand molecules occurs. Thus, infection via the normal route, e.g. for E. coli via wild type pili, is prevented by a modification to the host microorganism to prevent the display of a given type of surface molecule so that the only surface molecules of that type that are displayed are those fused to the receptor or ligand.
In a preferred aspect, the host microorganism expresses and displays modified ligand molecules, with the replicable genetic units expressing the candidate receptor molecules. Thus, this provides a method for selecting specific ligand and receptor encoding sequences wherein:
(a) the expression of a surface molecule encoding sequence is combined with a ligand encoding sequence, or a sequence encoding a functional fragment thereof, so that the host microorganism expresses modified ligand molecules on its surface;
(b) infecting, or in other ways transferring DNA to, the modified host organism of step (a) with a genetically modified replicable genetic unit capable of expressing a receptor, or a functional fragment thereof, fused to a surface protein of the replicable genetic unit; and,
(c) selecting infected host organisms containing specific ligand and receptor encoding sequences or sequences encoding functional fragments thereof.
In the above aspects, the method of the invention optionally includes the additional step of:
(d) isolating the nucleic acid sequences encoding the ligand or receptor molecules.
Conveniently, the isolation can be achieved by associating different selection markers (e.g. different antibiotic resistance markers) with the nucleic acid sequences encoding the ligand and receptor molecules. Thus, the host microorganisms containing nucleic acid sequences encoding a specific binding pair can be selected by growing them in the presence of both antibiotics. After this, the vectors containing nucleic acid encoding the ligand and the receptor can be separated from each other by omitting one of the antibiotics from the growing medium.
In different embodiments, this method can be used to screen:
(a) microorganisms displaying one type of ligand or receptor molecule against libraries of replicable genetic units displaying different receptor or ligand molecules;
(b) libraries of microorganisms displaying different ligand or receptor molecules against replicable genetic units displaying one type of receptor or ligand molecule; or,
(c) libraries of microorganisms displaying different ligand or receptor molecules against libraries of replicable genetic units displaying different receptor or ligand molecules.
In the above aspects, the host microorganisms do not express the normal (wild type) surface molecules that are used to display ligand or receptor molecules as fusions on its surface. This means that the replicable genetic units used to display candidate binding partners of the ligand or receptor molecule will only infect the microorganisms when binding between the ligand or receptor and a binding partner takes place, i.e. infection via the normal surface molecule of the microorganism is not possible as they are not displayed.
Thus, in one embodiment, the host microorganism is an E. coli strain in which the F episome is mutated in the traA gene that builds up pilin molecules, but which contains the other genes required for infection by bacteriophages, e.g. traL, traE, traY etc. Accordingly, these strains of E. coli are Fxe2x88x92, that is they do not produce functional pili, except when transformed with vectors comprising nucleic acid encoding fusions of the pilin and the ligand or receptor molecules. This allows wild type bacteriophages (e.g. M13, fd etc) displaying the candidate binding partners attached to a phage surface protein (e.g. pIII or pVIII protein) to be used, as the bacteriophage will only selectively infect those E. coli cells binding to the phage via the specific interaction of the binding partners. As the E. coli do not have wild type pili, this prevents the bacteriophage from infecting the E. coli unselectively. Thus, the present invention does not rely on engineering the replicable genetic units to limit their infectivity, and so obtain selectivity of DNA transfer, helping to avoid the difficulties that can be encountered in generating large libraries with deleted bacteriophages, such as pIII deleted M13 phage.
In embodiments of the invention that use the pili of the microorganism to display the ligand or receptor molecules, surprisingly good selectivity is obtained when the ligand or receptor nucleic acid is expressed as a fusion at the carboxy terminus of the pili.
Preferably, the vectors comprising the nucleic acid encoding the ligand and receptor molecules have different origins of replication (pME1, p15A, PSC101) so that both vectors can be stably maintained when transferred to the host microorganism.
In the method, steps (a) and (b) can be simultaneous or sequential. However, conveniently, the host microorganism will be cultured so that they display the ligand or receptor molecules, prior to infection with the replicable genetic units.
Accordingly, the present invention provides a method for selection of nucleic acid sequences encoding ligand and receptor molecules capable of specific binding to each other. The designed system enables the passage of DNA over cell membranes and is denoted xe2x80x9cCellular Linkage of ligand-receptor Affinity Pairsxe2x80x9d (CLAP). The DNA which is translocated from the replicable genetic unit over the cell membrane is denoted xe2x80x9cmembrane passage DNAxe2x80x9d (mp-DNA) and this genetic information outside the cell can be defined, constructed, designed and developed using recombinant DNA technology. The type of specific DNA, which is translocated over the cell membrane, is determined by the genetic information, DNA, inside the cell. This genetic information inside the cell can be defined, constructed, designed and developed using recombinant DNA technology and is denoted xe2x80x9ctranslocation determining DNAxe2x80x9d (td-DNA). Thus, in a preferred embodiment, this selectivity of DNA transfer is achieved using a strain of E. coli that have a mutation in the F episome so that they do not display functional pili. This means that when they are transformed with a vector comprising nucleic acid encoding a ligand or receptor for expression as a fusion with the pili, only these modified pili are displayed on the E. coli surface.
Based on the capability of specific td-DNA to determine the specific type of mp-DNA which is to be translocated into cells, the CLAP system can be used to construct a genetic library of td-DNA to determine the translocation of specific mp-DNA from a genetic library of mp-DNA. Thus, it is possible, in one single reaction, to mix a replicable genetic unit or units which encode genetic library/libraries of td-DNA and a genetic library/libraries of mp-DNA and these libraries encode proteins/peptides which are expressed from the genetic library/libraries. The individual td-DNA in individual cells will determine the translocation of individual mp-DNA into each cell.
Thus, the corresponding pairs of td-DNA and mp-DNA will be linked in the same cell. Individual td-DNA and mp-DNA can be separated and isolated using selection markers and therefore both the td-DNA and mp-DNA from the same cell can be separated, analyzed, defined, redesigned and reconstructed. The possibility to screen two gene libraries against each other in one reaction step allows the selection of ligand-receptor pairs without information of the individual clones building up the ligand-receptor pair. The td-DNA can be constituted of genes encoding proteins or peptides of various sizes. Individual cells encode specific proteins/peptides defined by the td-DNA in the cell and this protein or peptide is exposed on the cell surface. These exposed proteins/peptides can be constructed as mp-DNA and displayed on the surface of a replicable genetic unit. These proteins/peptides can be viewed as receptors and can then interact with ligand molecules exposed, encoded by the td-DNA, on the cell surface. When such interactions occur the ligand-receptor interaction mediates the passage of receptor DNA (mp-DNA) into the cell.
In one embodiment, the molecules on the cell surface are receptors and the molecules on the replicable genetic unit are ligands. A td-DNA can consist of a cDNA-library and the mp-DNA can consist of an antibody fragment gene library, where antibody fragment specificities can be selected against different protein/peptides ligand. The td-DNA can also consist of DNA encoding one type of peptide which can bind biotin and biotinylated chemical compounds. The biotin-peptide interaction allows the immobilization of biotinylated chemical compounds on the cell surface and these immobilized compounds can function as ligands/receptors for determining the translocation of mp-DNA encoding receptors/ligands. Antibody fragments with affinity to the immobilized chemical compound can be selected from a genetic library encoding different antibody specificities/affinities. The mp-DNA encoding the specificity/affinity of the selected antibody fragment is then replicated inside the cell, which displayed the biotin binding peptide binding to the biotinylated chemical compound. Other types of modifications of the chemical compounds can be used, which bind the biotin binding peptide and other types of peptides can be displayed on the cell surface which bind the modified chemical compound.
In a further aspect, the present invention provides a kit containing vectors for use in the methods described herein.
In this aspect, a preferred kit comprises:
(a) a host microorganism modified so that it does not display a wild type surface protein;
(b) a vector encoding said surface protein and having restriction sites for the insertion of nucleic acid encoding ligand or receptor molecules, or functional fragments thereof, so that when transformed into the host microorganism, the surface molecule and the ligand or receptor molecule are expressed as a fusion and displayed on the surface of the microorganism;
(c) a bacteriophage having a site for insertion of nucleic acid encoding candidate binding partners to the ligand or receptor so that the binding partners are expressed displayed on the surface of the bacteriophage as a fusion with a surface protein of the bacteriophage.
The above method can be used in a variety of applications, inter alia:
(1) cDNA libraries derived from, e.g. genome sequences, human or other tissue, can be cloned into the fusion vector and subsequently transfected into the microorganism host. Thus, by the way of expressing cDNA derived molecules as fusion proteins to pilin, we can use these cells as expressors of ligands. Subsequently, bacteriophages displaying antibody fragments derived from immunised or naive B cells from human or other origin will be formed and allowed to interact with the host microorganism expressing cDNA derived molecules. The infection event takes place and is mediated only by specific receptor-ligand interactions found between microorganisms and bacteriophages. The genetic information for each ligand and receptor pair can then be isolated.
(2) cDNA libraries derived from cancer patients and differentially PCR selected against normal tissue can be bacterially displayed, as described above. In the same manner, antibody libraries displayed on bacteriophages, derived from normal patients or cancer patients, are allowed to interact with the cDNA displaying microorganisms and infection is again mediated by specific receptor-ligand interactions.
(3) cDNA derived from allergen encoding gene sequences can be bacterially displayed as described above. In the same manner, antibody libraries displayed on bacteriophages derived from normal or atopic patients are allowed to interact with the cDNA displaying microorganisms and infection is again mediated by specific receptor-ligand interactions.